The neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20 one (3alpha, 5alpha-P or allopregnanolone) is a potent positive modulator of GABA receptors. Recent results from years 01-04 of the previous Center application suggest that genotypic differences in the modulatory effect of 3alpha,5alpha-P on EtOH withdrawal severity may reflect a balance between the change in sensitivity of the GABAa receptors to 3alpha, 5alpha-P that are occurring concomitantly during EtOH withdrawal. These studies were conducted in two different animal models of ethanol (EtOH) withdrawal severity [i.e., DBA/2 (D2) and C57BL/6(B6) inbred strains and the selectively bred Withdrawal Seizure-Prone and-Resistant lines]. In addition, gene mapping studies in BXD Recombinant Inbred strains found a significant genetic correlation between chronic EtOH withdrawal severity and a region of chromosome 13 (combined p=0.00008) in which the murine gene for the enzyme 5alpha-reductase-1 (Srd5alpha1). Since 5alpha-reductase is the rate-limiting enzyme in the biosynthesis of 3alpha, 5alpha-P, we hypothesize that Srd5a1 represents a candidate gene for differences in EtOH withdrawal severity in mice derived form the B6 and D2 inbred strains. Because male and female mice differ in endogenous levels of 3alpha, 5alpha-P (i.e., female > male) and in EtOH withdrawal severity ( female <male), studies related to Specific Aims 1 and 2 will determine whether male and female B6 and D2 mice differ in Srd5a1 sequence and whether the genotypes differ in enzyme activity and/or gene expression of Srd5a1 following exposure to chronic EtOH. The physiological consequence of 5alpha-reductase inhibition or deletion on EtOH withdrawal severity will be examined in Specific Aims 3 and 4. Therefore, the combined use of behavioral, biochemical, genetic and molecular strategies will determine the importance of 3alpha, 5alpha-P as a neuromodulator of EtOH withdrawal severity by assessing Srd5a1 as a candidate gene for genetic differences in EtOH withdrawal severity. These goals are consistent with the two main themes of the Center, which are to identify genes and to explore mechanisms underlying EtOH neuroadaptation. A longer-term goal of this research is to gain information that would help in the development of new strategies for the treatment of alcohol dependence. As recent findings emphasize the therapeutic potential of neurosteroid treatment during alcohol withdrawal, the studies proposed in Component #5 will determine whether neurosteroid biosynthesis represents a target for therapeutic intervention in the treatment of alcohol dependence and withdrawal.